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Magneto/Optical Annexin V, a Multimodal Protein Eyk A. Schellenberger,†,‡ David Sosnovik,† Ralph Weissleder,† and Lee Josephson*,† Center for Molecular Imaging Research, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, Massachusetts 02129, and Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115. Received April 14, 2004; Revised Manuscript Received June 15, 2004
Multimodal proteins, or proteins labeled with both fluorescent and magnetic reporter groups, can be used in a wide range of applications including FACS or fluorescence microscopy, MRI and or nearinfrared based optical imaging, or to fractionate cells by magnetic cell sorting. A problem with multimodal proteins, however, is the need to maximize bioactivity, often achieved by minimizing the number of modification points of the protein, while attaching fluorescent and magnetic labels. Here we describe the synthesis of a magneto/optical form of annexin V, achieved by reacting the aminoCLIO nanoparticle with Cy5.5 and SPDP, to produce a fluorescent, sulfhydryl reactive nanoparticle. A single reactive sulfhydryl group was added to annexin V by reaction with SATA that preserved the protein’s ability to bind apoptotic Jurkat T cells. Reacting SATAylated annexin V with an SPDP activated nanoparticle yielded Anx-CLIO-Cy5.5, a magneto/optical form of annexin V. The binding of Anx-CLIO-Cy5.5 was specific for apoptotic Jurkat T cells and had an EC50 of 3.66 nM. This was comparable to the strength of the interaction of unmodified annexin V with apoptotic cells, measured as the displacement of FITC-annexin by annexin V (2.4 nM). Our conjugation strategy preserves the strength of the interaction between annexin V and apoptotic cells, while yielding a probe, Anx-CLIOCy5.5, that is readily detectable by standard MR imaging or NIRF optical methods.
INTRODUCTION
Programmed cell death or apoptosis plays a crucial role during development (embryogenesis), for the maintenance of cellular homeostasis in the adult, and in the pathology of a wide range of diseases. Apoptosis is a central feature in diseases such as AIDS, neurodegenerative disorders (e.g., Alzheimer’s disease), in myelodysplastic syndromes such as aplastic anemia and thalassemia, in progressive heart failure, for chronic hepatitis, and for transplant rejection (1-3). As cells proceed along pathways to apoptosis, phosphatidylserine (PS), a lipid normally facing the cytoplasm, flips and faces the extracellular fluid (4, 5). The human protein annexin V (36 kDa) binds PS strongly and specifically, which is a reflection of a biological role as an anticoagulant (6, 7). The binding of annexin V to PS is an attractive method for determining apoptosis in organs and tissues since PS is expressed early in the commitment to apoptosis (4, 8) and is a feature of apoptotic cells regardless of how apoptosis is induced (7). The ability of annexin V to recognize PS has led to the development of fluorescent or magnetic annexins that are useful in a variety of applications. Annexin V labeled with FITC is a widely used for identifying apoptotic cells by FACS (9, 10), while a Cy5.5 labeled annexin V has been used for the NIRF/optical imaging of chemotherapyinduced apoptosis (11, 12). Magnetic annexin V’s are useful reagents for the removal of apopotic cells (13, 14). * To whom correspondence should be addressed. Center for Molecular Imaging Research, Massachusetts General Hospital, Building 149, 13th Street, Room 5406, Charlestown, Massachusetts 02129. Fax: (617) 726-5708. E-mail: ljosephson@ partners.org. † Massachusetts General Hospital, Harvard Medical School. ‡ Present address: Department of Radiology, Charite ´ Universita¨tmedizin Berlin, Campus Charite´ Mitte, Berlin, Germany.
Here we describe the development of a magneto/optical annexin V that retains its binding activity for apoptotic cells, while being detectable by both fluorescent and magnetic resonance imaging modalities. Unlike earlier modified annexin V’s, which are based on the direct attachment of fluorochromes to annexin V, or the conjugation of annexin V to magnetic particles, the magneto/ optical annexin V is based on the attachment of annexin V to a nanoparticle that is both magnetic and fluorescent. The multimodal annexin V can be used with cell-based, fluorescence methods to identify apoptotic cells (FACS or fluorescent microscopy), with small animal optical imaging, or by MRI. MATERIALS AND METHODS
Synthesis of Magneto/Optical Annexin V. The synthesis of magneto/optical annexin V consisted of three steps. First, the amino-CLIO (cross-linked iron oxide) nanoparticle was labeled with Cy5.5 and activated with SPDP, to yield a compound termed 2PySS-CLIO-Cy5.5. Second, annexin V was reacted with SATA in a manner that preserved its affinity for apoptotic cells Third, SATAylated annexin V was reacted with 2PySS-CLIO-Cy5.5 to yield the multimodal nanoparticle Anx-CLIO-Cy5.5. Amino-CLIO was prepared as described (15, 16). To 3.5 mL of amino-CLIO (∼10 mg Fe) we added 2.36 mL of 0.1 M sodium bicarbonate, pH 8.5. The buffered nanoparticle was then added to 1.75 mg of the monofunctional N-hydroxysuccinimde ester of Cy5.5 (Amersham-Pharmacia, Piscataway, NJ) dissolved in 0.175 mL of DMSO and incubated for 2 h at room temperature. We then added 0.6 mL of 120 mM SPDP (N-succinimidyl 3-(2-pyridyldithio) propionate) in DMSO and incubated for an additional 2 h. Unreacted Cy5.5 and SPDP were removed by gel filtration (Sephadex G-25 column equilibrated with 0.02 M Na citrate, 0.02 M Na borate, 0.9 M NaCl, pH 8.5). The number of Cy5.5 per nanoparticle was
10.1021/bc049905i CCC: $27.50 © 2004 American Chemical Society Published on Web 08/19/2004
Magneto/Optical Annexin V
determined from the concentrations of iron (16) and Cy5.5 dye, the later being determined spectrophotometrically (E678 ) 250 000), and by assuming 2064 iron atoms per nanoparticle (17). The number of 2PySS groups per nanoparticle was determined after the release of 2-pyridine-thione (2-PT) with dithiothreitol (18). The resulting nanoparticle, termed 2PySS-CLIO-Cy5.5, was stored at 4 °C until needed. Annexin V was obtained from Theseus Imaging Corporation (Worcester, MA). To 1 mL of annexin V (3 mg) was added 600 µL of 0.1 M sodium bicarbonate, pH 8.6, and 12 µL of 10.8 mM SATA (N-succinimidyl 3-(2pyridylthio)propionate, S-acetylthioactetate) dissolved in DMSO. After 1 h at room temperature under argon, 220 µL of a solution of 0.01 M hydroxylamine, 0.01 M EDTA (pH 8) was added. The mixture was incubated for 40 min at room temperature under argon, and unreacted SATA separated from annexin V by spin separation (Biogel P6 columns equilibrated with 0.1 M sodium bicarbonate, pH 8). We then added 200 µL of 2Py-SS-CLIO-Cy5.5 to the SATAylated annexin V and allowed the mixture to stand overnight at room temperature under argon. Unreacted annexin V was removed by gel filtration (Sephadex G-150 equilibrated in 0.02 citrate 0.15 M NaCl, pH 8). Protein content of the resulting nanoparticle was determined by the BCA method (Pierce Chemical, Rockford, IL). Size of the nanoparticle was determined by light scattering using a Zetasizer (Malvern Instruments, Westboro, MA). Relaxivities were measured at 0.47 T and 40 °C using a relaxometer (Bruker Instruments, Braintree, MA). Interaction of Anx-CLIO-Cy5.5 with Apoptotic Cells. Jurkat T cell cells (Clone E6-1, ATCC #TIB-152) grown in RPMI 1640 medium with 10% fetal bovine serum (Vitacell #30-2021, ATCC, Manassas VA) changed every 2 or 3 days were used in all studies. Apoptosis was induced by the addition of 7 µL of camptothecin (1 mM in DMSO) per milliliter of culture medium for 5-6 h at 37 °C. Induction of apoptosis was verified by staining with propidium iodide and FITC-annexin V using a calcium-containing binding buffer (1.8 mM CaCl2, 10 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, pH 7.4). The cells were then analyzed with a FACS-Calibur cytometer (Becton Dickinson, San Jose, CA). Competition Experiments. The dye-annexin V’s we employed (Cy5.5 or FITC) had one mole of dye per mole of annexin V and were prepared as described (11, 12). To assess the bioactivity of annexin V SATAylated to various extents, the ability to block the binding of FITCannexin V to apoptotic cells was determined by FACS (11). Camptothecin-treated Jurkat T cells (104-105 cells) were incubated with different SATAylated annexin V’s (1.0 µg in 200 µL binding buffer) for 5 min at room temperature. FITC-annexin V (0.1 µg) was then added for an additional 10 min at room temperature. Binding of FITC-annexin V to apoptotic cells was determined from the medians of the highest peaks from the FACS analysis as described (11). The relative FITC-annexin V fluorescence (Figure 1) was obtained by dividing the median value obtained of SATAylated annexin V’s by that median value with no SATAylated annexin, that is by normalization to the binding in the absence of a SATAylated annexin V. IC50 values of Anx-CLIO-Cy5.5, annexin V, and Cy5.5-annexin V were determined by the ability to displace FITC-annexin V (4.1 nM or 0.03 µg/ 500 µL) in the assay above. The direct binding of AnxCLIO-Cy5.5 to apoptotic cells was assessed by determining the relative cell associated fluorescence in the Cy5.5 channel of the apoptotic cell fraction for campto-
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Figure 1. Binding of SATA-annexin V conjugates to camptothecin treated Jurkat T cells. Active SATA-annexin V's displace FITC-annexin V to yield a low Relative Binding by FACS. Inactive SATA-annexin V's, or no annexin V, fail to displace yielding a high Relative Binding. Relative binding is obtained by normalizing to the median fluorescence of the apoptotic cell fraction by FACS. Table 1. Properties of Anx-CLIO-Cy5.5 moles of annexin V per CLIO moles of Cy5.5 dyes per CLIO size [nm] R1 [mM-1 s-1] R2 [mM-1 s-1]
3.5 1.8 50 19 48
thecin-treated Jurkat T cells over a range of probe concentrations. The data were fitted to sigmoidal doseresponse curves to obtain EC50’s, IC50’s, values of the Hill coefficient (n), and the correlation coefficient (R2), indicating the goodness of fit, using Prism 4 (GraphPad Software, San Diego, CA). Optical and Magnetic Imaging With Anx-CLIO-Cy5.5. Jurkat T cells were incubated with Anx-CLIO-Cy5.5 (1 µg Fe/mL) for 10-15 min and washed twice with calcium binding buffer. Cell pellets in 300 µL tubes were prepared by centrifugation and imaged for surface reflectance fluorescence as described (19). MR imaging was performed on a 4.7 T Bruker BioSpin scanner. Transverse relation times (T2’s) were determined by fitting signal intensity data (TE ) 10-200 ms and TR ) 2000 ms) to the spin echo signal intensity equation (20). A T2 weighted image was also taken at TE ) 50/TR ) 2000 (Figure 4). RESULTS
Synthesis of Magneto/Optical Annexin V. The aminoCLIO nanoparticle was reacted with the NIRF dye Cy5.5 and then with the bifunctional reagent SPDP, followed by a single gel filtration step to remove all low molecular weight impurities. The nanoparticle, 2PySS-CLIO had 71 2PySS groups per nanoparticle, assuming 2064 Fe/ nanoparticle (17). 2PySS-CLIO-Cy5.5 could be stored at 4 °C and is a general reagent for converting sulfhydrylbearing proteins in to magneto/optical probes. The properties of the dual modality nanoparticle Anx-CLIO-Cy5.5 are summarized in Table 1. The sensitivity of annexin V to modification of its amino groups by SATA using a FACS displacement assay is shown in Figure 1. The assay indicates that the attachment of one SATA per mole of annexin V does not affect binding to apoptotic cells. On the basis of these results, annexin V with on average one SATA per mole was reacted with the SPDP-activated amino-CLIO. The resulting nanoparticle had 3.5 annexin V’s per nanoparticle with R1 and R2’s of 19 and 48 mM-1 s-1, respec-
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Figure 2. Dual wavelength FACS analysis with Anx-CLIO-Cy5.5 and FITC-annexin V. Untreated cells (A) and camptothecintreated cells (B) are shown. Results from (A) and (B) are tabulated in (C).
tively, and a diameter of 50 nm by light scattering, which are values similar to CLIO nanoparticles functionalized with other types of biomolecules (15, 21, 22). Binding of Magneto/Optical Annexin V, Anx-CLIOCy5.5, to Apoptotic Cells. Dual wavelength FACS with FITC-annexin as a reference is an excellent method for examining the specificity of probes such as Anx-CLIOCy5.5 for apoptotic cells. Anx-CLIO-Cy5.5 and FITCannexin V bound in a similar manner to individual cells with populations of normal (Figure 2A) or camptothecintreated cells (Figure 2B). The binding of probes to cells was in a similar manner at least 95% of the time (Figure 2C). Thus, the conjugation of annexin V to CLIO preserves the specificity of the annexin V interaction with apoptotic cells. We next examined the strength of the interaction of Anx-CLIO-Cy5.5 with apoptotic cells as shown in Figure 3, using both direct binding and displacement assays. Using the direct binding assay for Anx-CLIO-Cy5.5, we obtained an EC50 of 3.66 nM per mole of annexin V, which can be compared to values expressed as dissociation constants as low as 0.1 nM (23) or as high as 7 nM (24). The conjugation of annexin V to Anx-CLIO-Cy5.5 preserves the strength of the interaction between annexin V and apoptotic cells. However, when the ability of AnxCLIO-Cy5.5 to displace FITC-annexin was examined, an IC50 of 39 nM was obtained which was higher than the IC50 for annexin V (2.40 nM) or Cy5.5-annexin (7.8 nM). The multimodal probe Anx-CLIO-Cy5.5 thus interacts with apoptotic cells specifically (Figure 2) and with an affinity similar to native annexin V (3.66 versus 2.40 nM). However, the Anx-CLIO-Cy5.5 nanoparticle, and annexin V protein may interact with the phosphati-
dylserine in a somewhat different fashion, based on their differing ability to displace FITC-annexin (Anx-CLIOCy5.5 IC50 ) 39 nM; annexin V IC50 ) 2.4 nM). The presence of 3.5 annexin V’s presented on a single 50-nm nanoparticle may bind PS somewhat differently from the 36 kDa annexin V molecule. Optical and Magnetic Imaging With Anx-CLIO-Cy5.5. We next examined the detection of cells labeled with AnxCLIO-Cy5.5 by both NIRF and MR imaging modalities. A high signal intensity NIRF signal was observed with apoptotic but not normal cells (Figure 4B) and a corresponding decrease in signal intensity (darkening) of cells was obtained with a T2 weighted MR image (Figure 4C). T2’s were 76 ms for the control and 37 ms for the camptothecin-treated cells. DISCUSSION
Multimodal, or fluorescent and magnetic, nanoparticles have numerous advantages over single modality based probe designs (25-27). During the research phase of probe development, fluorescence can be used with FACS or fluorescence microscopy to characterize the specificity of interactions with cells or study intracellular processing. Small animal NIRF based imaging for assessing probe targeting is often more convenient and cheaper than MRI. Fluorescence permits the use of multiple, optically distinct fluorochrome probes in conjunction with FACS, microscopy, or animal imaging devices (19, 25). However, despite the rapid advances in small animal optical imaging technology and possible translation of small animal to human imaging (28, 29), the development
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Figure 3. Anx-CLIO-Cy5.5 binding to apoptotic Jurkat T cells. (A) Direct binding assay where Anx-CLIO-Cy5.5 binding is obtained by normalizing the median fluorescence of apoptotic T cells (camptothecin treated) obtained by flow cytometry. (B) Competition assay showing the displacement of FITC-Annexin V by Anx-CLIO-Cy5.5, Cy5.5-annexin V or annexin V. (C) Results from (A) and (B) are tabulated.
Figure 4. Multimodal imaging of Anx-CLIO-Cy5.5 binding to apoptotic cells. (A) White light image of pellets of control cells (left) and camptothecin-treated cells (right). Control cells (left) were 7.2% apoptotic, while camptothecin-treated cells were 55.3% apoptotic by FACS using FITC-annexin V. (B) NIRF image of the tubes from (A) show high fluorescence of apoptotic cells (C) MRI of cells in (B) shows camptothecin-treated cells (right) had a lower signal intensity, i.e., were dark, compared with the untreated cells.
and distribution of NIRF based clinical imaging systems lie well in the future while MRI is an established imaging modality. The strategy we have employed for the design of AnxCLIO-Cy5.5 yields a multimodal (magnetic and fluores-
cent) form of annexin V that differs from earlier fluorescent, radioactive, or magnetic annexin V’s (10, 11, 30, 31). First, we preserved the PS binding activity of annexin V by optimizing the reaction between annexin V and SATA (Figure 1). Annexin V loses its PS binding activity if more than 1 lysine per mole is modified by either SATA or fluorochromes (32), and appears to be highly sensitive to modification. We then attached SATAylated annexin V (36 kDa) to the SPDP-activated CLIO, which was about 1000 kDa (or about 30 nm). The nanoparticle had 2064 Fe atoms and 1.8 Cy5.5 dyes per nanoparticle. The resulting Anx-CLIO-Cy5.5 probe was highly active compared to the parent annexin V (Figure 3). Anx-CLIOCy5.5 is a PS-binding magneto/optical form of annexin V that is about 1000 kDa rather than 36 kDa, featuring 590 Fe atoms per annexin V (2064/3.5) for MR detection and 0.5 Cy5.5 molecules per annexin V (1.8/3.5) for detection by fluorescence. The low molecular weight, predominant renal elimination, and short blood half-life of the 36 kDa form of annexin V may limit its ability to bind its target PS. Annexin V shows predominant renal elimination with a blood half-life 24 min in humans (33) and less than 10 min in mice (34, 35). Radiolabeled PEGylated annexin V’s have been developed in an effort to increase blood half-life and improve PS targeting (36), but based on the sensitivity of annexin V to modification, the attachment of PEG and DTPA to a single annexin V poses a considerable risk of inactivating the PS binding activity. Anx-CLIO-Cy5.5 is too large to undergo renal elimination and may have a longer blood half-life than annexin V. Magneto/optical nanoparticles have been successfully used in several in vivo imaging applications. A fluorescent nanoparticle was used to image the cathepsin B activity of lymph nodes in live mice by simple surface reflectance and to obtain high-resolution MR images of nanoparticle disposition (27). We have shown that intra-
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venously injected Cy5.5 labeled CLIO is internalized by microglia cells, and can be used as a preoperative MR contrast agent for the visualization of brain tumor in an animal model (26). The agent also can be seen by NIRF surface reflectance after removal of the cranium, and provides a method of determining tumor margins intraoperatively. Multimodal probes utilizing dextran rather than nanoparticle carriers have employed gadolinium and (fluorochrome), and have been used to study stem cell migration (37, 38). The addition of a fluorochrome to the magnetic nanoparticle permits FACS analysis or fluorescent microscopy to determine the disposition of the MR contrast agent. The near-infrared fluorescence of Anx-CLIO-Cy5.5 is distinct from many fluorochromes such as fluorescein or Alexafluor 488, which can be used in colabeling studies to characterize the subpopulation of cells binding the multimodal nanoparticle by FACS analysis, without spillover or compensation. With tissue sections and fluorescent microscopy, there is considerably less background with NIRF probes because mammals do not synthesize near-infrared fluorochromes. Other approaches to visualizing nanoparticles, such as iron staining or histochemical methods, require specific staining reagents and can suffer from higher backgrounds. The fluorescent and magnetic character of Anx-CLIOCy5.5 is not only well suited to the determination of probe behavior in vitro and preclinically, but also offers multiple pathways to the development of a clinical diagnostic agent. First, the fluorochrome can be dropped and an annexin V-polymer coated superparamagnetic iron oxide could be used as an MR contrast agent. Polymer-coated iron oxides are used as MR contrast agents for the liver, lymph nodes, and gastrointestinal tract, and are also used to treat anemia. Although optical imaging is undergoing rapid development (39, 40), MRI currently has the advantages of wide clinical availability and the ability to visualize probe accumulation deep within the human body. Second, the targeting biomolecule, in this case annexin V, and NIRF fluorochromes might be attached to a fluorochrome loaded polymeric molecules such as dextran, to yield a high molecular weight form of annexin V for optical imaging. Annexin V at 36 kDa undergoes renal elimination (33, 35), while higher molecular weight forms may have substantially different and, in some applications, improved pharmacokinetics. The ability to image apoptotic cells in vivo would likely be of significant value in both the diagnosis and treatment of many diseases and treatments. However, in many organs only a small fraction cells undergo apoptosis at any given moment, and thus probes with an optimized conjugation and labeling chemistry may be needed. Future studies will determine the ability of Anx-CLIOCy5.5 to detect apoptosis as the percentage of apoptotic cells by MRI and NIRF imaging methods and compare the 36 kDa Dye-annexin V conjugates and Anx-CLIOCy5.5 nanoparticle as optical imaging agents. ACKNOWLEDGMENT
Work was supported by NIH Grants RO1CA86782, P50CA86355, R24 CA92782, and CA91807. E.A.S. was funded by a grant from the “Deutsche Akademie der Naturforscher und Mediziner Lepoldina.” D.S. was supported by a Research Fellow Award from the Radiological Society of North America. LITERATURE CITED (1) Thompson, C. B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456-1462.
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BC049905I
